Novel immunomodulating compounds

ABSTRACT

The present invention relates to a novel heterocyclic compound, a pharmaceutical composition comprisining said compound, a method and use of said compound for clinical treatment of medical conditions which may benefit from immunomodulation, e.g. rheumatoid arthritis, multiple sclerosis, diabetes, asthma, transplantation, systemic lupus erythematosis and psoriasis. More particularly the present invention relates to novel heterocyclic compounds, which are CD80 antagonists capable of inhibiting the interactions between CD80 and CD28.  
     A method of screening compounds for their ability of inhibiting ligand-induced co-stimulatory receptor internalisation pathways in immune competent human cells is described. Said immune competent human cells are incubated at conditions capable of inducing co-stimulatory receptor internalisation in the presence of at least one test compound and the suppression of the ligand-induced co-stimulatory receptor internalisation determined.  
     There is also described a kit for use in such a method, as well as an immunoregulatory drug capable of blocking down-modulation of a ligand-induced receptor.

FIELD OF THE INVENTION

[0001] The present invention relates to immunomodulating compounds,pharmaceutical compositions comprising said compounds, use of saidcompound as well as a method for treatment of medical conditions whichbenefit from immunomodulation, wherein said compounds are administered.

BACKGROUND OF THE INVENTION

[0002] The immune system possesses the ability to control thehomeostasis between the activation and inactivation of lymphocytesthrough various regulatory mechanisms during and after an immuneresponse. Among these are mechanisms that specifically inhibit and/orturn off an immune response. Thus, when an antigen is presented by MHCmolecules to the T-cell receptor, the T-cells become properly activatedonly in the presence of additional co-stimulatory signals. In theabsence of accessory signals there is no lymphocyte activation andeither a state of functional inactivation termed anergy or tolerance isinduced, or the T-cell is specifically deleted by apoptosis.

[0003] One such co-stimulatory signal involves interaction of CD80 onspecialised antigen-presenting cells with CD28 on T-cells, which hasbeen demonstrated to be essential for full T-cell activation. (Lenschowet al. (1996) Annu. Rev. Immunol., 14, 233-258).

PRIOR ART

[0004] In U.S. Pat. No. 4,312,870 compound A is disclosed as one ofseveral psychoactive compounds but without any biological data. Somerelated compounds are described by A. Carotti in Bioorganic & MedicinalChemistry 6 (1998) 389-399, and from data related to these compounds itis obvious that the carboxylic acid substituent greatly diminishesbiologic activity measured as affinity for the CNS benzodiazepinereceptor.

[0005] EP 0354693A1 (Boots) discloses immunomodulatory compounds ofgeneral structure B but does not include structures wherein R7 and/or R8are COOH or contain a COOH group.

[0006] Similarly EP 0354694A1 (Boots) discloses immunomodulatorycompounds of general structure C but no structures wherein R6 and/or R7are COOH or contain a COOH group are described.

[0007] Also, WO9111448 (Boots) discloses immunomodulatory compounds ofgeneral structure D but here are no structures wherein R7 and/or R8 andR8′ are COOH or contain a COOH group.

SUMMARY OF THE INVENTION

[0008] The present invention relates in a first aspect to a novelcompound having the general formula (I)

[0009] wherein X represents a bond or a group selected from substitutedor unsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ orC(O)—NH— (amino acid residue);

[0010] Y represents NR4, O or S;

[0011] R1 represents H, halo, CF₃, lower alkyl or lower alkoxy;

[0012] R2 and R4 represents independently H or lower alkyl; and

[0013] R3 represents H, halo, lower alkyl or lower alkoxy, wherein halois F, Cl or Br;

[0014] wherein lower alkyl represents saturated or unsaturated,straight, branched or cyclic alkyl groups having 1-6 carbon atoms; and

[0015] wherein lower alkoxy represents saturated or unsaturated,straight, branched or cyclic alkoxy groups having 1-6 carbon atoms,

[0016] with the proviso that R2 is not H, when X is a bond and Y is NHand R3 is H,

[0017] or pharmaceutically acceptable salts thereof.

[0018] In one preferred embodiment of the invention the compound X is abond and in another preferred embodiment Y is NH.

[0019] In further embodiments the compound is selected from the groupcomprising

[0020]{[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy}-aceticacid,

[0021]N-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicacid,

[0022]4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-butyricacid,

[0023]{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy}-aceticacid,

[0024]4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-2-phenyl-butyricacid,

[0025]N-[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicacid,

[0026]2-{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methyl}-benzoicacid,

[0027]2-Chloro-4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0028]4-(6,8-Dimethyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0029]4-(8-Methoxy-6-methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0030]4-(6,8-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0031]4-(7,9-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0032]4-(6-Methyl-3-oxo-8-trifluoromethyl-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0033]4-(7,9-Dichloro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0034][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-propionicacid,

[0035][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-acetic acid,

[0036] 4-(4-Methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzoic acid,

[0037] 4-(3-Oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)benzoic acid,

[0038]4-(5-Methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,

[0039] 2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-3-phenyl-propionic acid, and

[0040] 2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-2-acetic acid.

[0041] In yet another embodiment said compound is a CD80 antagonist,capable of inhibiting the interaction between CD80 and CD28.

[0042] The present invention relates in a second aspect to a compound asset forth above for use as a medicament.

[0043] In one preferred embodiment said compound is used as a medicamentfor treatment of medical conditions chosen from the group comprisingrheumatoid arthritis, multiple sclerosis, diabetes, asthma,transplantation, systemic lupus erythematosis and psoriasis.

[0044] The present invention relates in a third aspect to a compound asset forth above for use as a prodrug, preferably in the form of anester.

[0045] The present invention relates in a fourth aspect to apharmaceutical composition comprising said compound as active ingredientin association with a pharmaceutically acceptable adjuvant, diluent orcarrier.

[0046] In one prefered embodiment said pharmaceutical composition isused for treatment of medical conditions chosen from the groupcomprising rheumatoid arthritis, multiple sclerosis, diabetes, asthma,transplantation, systemic lupus erythematosis and psoriasis.

[0047] The present invention relates in a fifth aspect to the use of acompound having the general formula (I)

[0048] wherein X represents a bond or a group selected from substitutedor unsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ orC(O)—NH— (amino acid residue);

[0049] Y represents NR4, O or S;

[0050] R1 represents H, halo, CF₃, lower alkyl or lower alkoxy;

[0051] R2 and R4 represents independently H or lower alkyl; and

[0052] R3 represents H, halo, lower alkyl or lower alkoxy,

[0053] wherein halo is F, Cl or Br;

[0054] wherein lower alkyl represents saturated or unsaturated,straight, branched or cyclic alkyl groups having 1-6 carbon atoms; and

[0055] wherein lower alkoxy represents saturated or unsaturated,straight, branched or cyclic alkoxy groups having 1-6 carbon atoms, orpharmaceutically acceptable salts thereof, for the manufacture of amedicament for treatment of medical conditions which benefit fromimmunomodulation.

[0056] In one embodiment said medical conditions are chosen from thegroup comprising rheumatoid arthritis, multiple sclerosis, diabetes,asthma, transplantation, systemic lupus erythematosis and psoriasis.

[0057] The present invention relates in a sixth aspect to a method fortreatment of medical conditions which benefit from immunomodulationcomprising administration of a therapeutically effective amount of acompound having the general formula (I)

[0058] wherein X represents a bond or a group selected from substitutedor unsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ orC(O)—NH— (amino acid residue);

[0059] Y represents NR4, O or S;

[0060] R1 represents H, halo, CF₃, lower alkyl or lower alkoxy;

[0061] R2 and R4 represents independently H or lower alkyl; and

[0062] R3 represents H, halo, lower alkyl or lower alkoxy,

[0063] wherein halo is F, Cl or Br;

[0064] wherein lower alkyl represents saturated or unsaturated,straight, branched or cyclic alkyl groups having 1-6 carbon atoms; and

[0065] wherein lower alkoxy represents saturated or unsaturated,straight, branched or cyclic alkoxy groups having 1-6 carbon atoms, orpharmaceutically acceptable salts thereof.

[0066] In one embodiment said medical conditions are chosen from thegroup comprising rheumatoid arthritis, multiple sclerosis, diabetes,asthma, transplantation, systemic lupus erythematosis and psoriasis.

[0067] In another embodiment said therapeutically effective amount perday is within the range of 0,001-10 mg/kg body weight, preferably withinthe range of 0,1-5 mg/kg body weight.

DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention relates to heterocyclic compounds, topharmaceutical compositions comprising them, and to methods and use ofsaid compounds for clinical treatment of medical conditions which maybenefit from immunomodulation, e.g. rheumatoid arthritis, multiplesclerosis, diabetes, asthma, transplantation, systemic lupuserythematosis and psoriasis. More particularly the present inventionrelates to heterocyclic compounds, which are CD80 antagonists capable ofinhibiting the interactions between CD80 and CD28.

[0069] According to the present invention it has been found thatcompounds of general formula I are CD80 antagonists. Compounds of thegeneral formula I inhibit the interaction between CD80 and CD28. TheCD80 antagonistic properties of the compounds of general formula I havebeen established in Surface Plasmon Resonance (BIAcore) experiments.

[0070] It is preferred that X in the general formula I is a bond and insuch cases other substituents, such as an adjacent meta-chloro, improvesactivity.

[0071] The compounds of the present invention may be in the acid formbut may also be in the form of pharmaceutically acceptable salts.

[0072] The compounds of the present invention may also be in the form ofprodrugs, especially esters with appropriate alcohols. Prodrugs can haveimproved pharmacokinetic and/or solubility properties.

[0073] Since the compounds of formula I are CD80 antagonists capable ofinterfering with the CD80-CD28 interaction they are useful for treatmentof inflammatory conditions and autoimmune diseases, e.g. rheumatoidarthritis, multiple sclerosis, diabetes, asthma, transplantation,systemic lupus erythematosis and psoriasis.

[0074] Effective quantities of the compounds of formula I are preferablyadministered to a patient in need of such treatment according to usualroutes of administration and formulated in usual pharmaceuticalcompositions comprising an effective amount of the active ingredient anda suitable pharmaceutically acceptable carrier. Such compositions maytake a variety of forms, e.g. solutions, suspensions, emulsions,tablets, capsules and powders prepared for oral administration, sterilesolutions for parenteral administration, suppositories for rectaladministration or suitable topical formulations. Conventional proceduresfor the selection and preparation of suitable pharmaceuticalformulations are described, for example, in “Pharmaceuticals—The Scienceof Dosage Form Design”, M. B. Aulton, Churchill Livingstone, 1988.

[0075] A suitable daily dose for use in the treatment of any of theconditions mentioned above is within the range of 0.001 mg/kg to 10mg/kg body weight, in particular within the range of 0.01 mg/kg to 5mg/kg body weight, depending upon the specific condition to be treated,the age and weight of the specific patient, and the specific patient'sresponse to the medication. The exact individual dosage, as well as thedaily dosage, will be determined according to standard medicalprinciples under the direction of a physician.

[0076] Various additives to enhance the stability or ease theadministration of the drug can be added to the formulation. Thepharmaceutical composition may also contain additional therapeuticallyuseful substances other than one or more compounds of the generalformula I.

[0077] The present invention is further illustrated by the followingnon-limiting experimental part.

[0078] Experimental Part

[0079] The compounds of general formula I may be prepared by the methodsdescribed below. The prior art patent documents cited above also includeuseful synthetic methods.

[0080] Thus, compounds of general formula I wherein Y=N and X is analkyl chain can be prepared as shown in the reaction below;

[0081] The starting material is available by known procedures (e.g. L.Savini et al, Bioorganic & Medicinal Chemistry 6 (1998) 389-399) and thereaction with hydrazine derivatives is performed by heating in asuitable solvent such as n-butanol. In cases where the acid isesterified under the reaction conditions, hydrolysis gives back theacid. Preparation of hydrazine derivatives was accomplished followingliterature procedures (Hunsberger et al, J. Org. Chem. 21 (1956) 394,395, 396. Harden F. A. et al, J. Med. Chem. 34 (1991) 2892-8).

[0082] Compounds wherein R4 is lower alkyl may be obtained by subsequentalkylation. If the alkylation results in ester formation, thecorresponding acid is easily obtained by hydrolysis.

[0083] The compounds of general formula I wherein Y=O and X is an alkylchain can be prepared according to various literature procedures (e.g.Ghosh C. K. Et al, Synthesis (1978) 779-781; Frogett J. A. et al, J.Chem. Research (S) (1997) 30-31). One synthetic route, when R2=Me, isdescribed below. The cyano group is subsequently hydrolysed to producethe corresponding acid.

[0084] The compounds of general formula I wherein Y=S and X is an alkylchain can be prepared as shown below using procedures described in theliterature (Donelly M. X. D. et al, J. Chem. Soc. Perkin Trans. 1 (1993)1729-1735; Lombardino J. G. et al, J. Med. Chem. 24 (1981) 830-834). Thefinal oxidation was accomplished by stirring in air.

[0085] The compounds wherein X is a bond may also be obtained from thecorresponding cyanide derivatives and the amino acid derivatives areprepared by condensation of the acid with the appropriate amino acid.

[0086] Compounds wherein X is NH—C(O)-(alkyl)- and NH—C(O)—CH₂—O—CH₂—can be prepared by reduction of the nitro group (L. Savini et al,Bioorganic & Medicinal Chemistry 6 (1998) 389-399) and subsequentacylation with the appropriate anhydride.

EXAMPLES

[0087] The following examples are intended to illustrate the inventionwithout restricting the scope thereof. Compounds were named usingAutonom 2.1 from Beilstein. NMR spectra were recorded on a Bruker ARX400 instrument. Coupling constants in the aromatic area are mostlyreferred to as singlets (s), doublets (d), triplets (t) in order toreflect the appearance of the NMR spectrum.

Example 1

[0088]{[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy)-aceticAcid

[0089] To a suspension of2-(3-amino-phenyl)-2,5-dihydro-pyrazolo[4,3-c]quinolin-3-one (0.050 g,0.18 mmol) in DMF (0.8 ml) diglycolic anhydride (0.025 g, 0.22 mmol) and4-dimethylaminopyridine (0.007 g, 0.05 mmol) were added. The clearsolution was stirred at room temperature and the product precipitatedduring the reaction. After 3 h water was added to the mixture. Theprecipitate was collected, washed with water and dried to yield{[3-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl-carbamoyl]-methoxy}-aceticacid (0.064 g): ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (1H, bs), 10.02 (1H,s), 8.69 (1H, s), 8.37 (1H, s), 8.18 (1H, d), 7.95 (1H, d), 7.62-7.70(2H, m), 7.49-7.55 (2H, m), 7.33 (1H, t), 4.19 (2H, s), 4.17 (2H, s);ESI MS m/z 393 (M+H⁺)

[0090] Using essentially the same procedure the following compounds wereprepared:

[0091]N-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicAcid

[0092]¹H NMR (400 MHz, DMSO-d₆) δ 9.98 (1H, s), 8.67 (1H, s), 8.18 (1H,d), 8.08 (2H, d), 7.58-7.70 (4H, m), 7.51 (1H, t), 2.47-2.57 (4H, m);ESI MS m/z 377 (M+H⁺).

[0093]4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-butyricAcid

[0094]¹H NMR (400 MHz, DMSO-d₆) δ 9.89 (1H, s), 8.67 (1H, s), 8.18 (1H,d), 8.08 (2H, d), 7.60-7.69 (4H, m), 7.51 (1H, t), 2.33 (2H, t), 2.25(2H, t), 1.75-1.83 (2H, m); ESI MS m/z 391 (M+H⁺).

[0095]{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy}-aceticAcid

[0096]¹H NMR (400 MHz, DMSO-d₆) δ 12.76 (1H, bs), 9.86 (1H, s), 8.68(1H, d), 8.19 (1H, d), 8.11 (2H, d), 7.61-7.69 (4H, m), 7.52 (1H, t),4.19 (2H, s), 4.15 (2H, s); ESI MS m/z 393 (M+H⁺).

[0097]4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-2-phenyl-butyricAcid

[0098]¹H NMR (400 MHz, DMSO-d₆) (contain some 3-phenyl regioisomer) δ9.86 (1H, s), 8.67 (1H, s), 8.18 (1H, d), 8.07 (2H, d), 7.58-7.69 (4H,m), 7.51 (1H, t), 7.20-7.38 (5H, m), 3.55 (1H, t), 2.10-2.30 (3H, m),1.90-2.02 (1H, m); ESI MS m/z 467 (M+H⁺).

[0099]N-[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicAcid

[0100]¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (1H, s), 8.68 (1H, s), 8.31 (1H,s), 8.17 (1H, d), 7.90 (1H, d), 7.62-7.70 (2H, m) , 7.48-7.55 (2H, m)7.30 (1H, t), 2.49-2.57 (4H, m) ESI MS m/z 377 (M+H⁺)

[0101]2-{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methyl}-benzoicAcid

[0102]¹H NMR (400 MHz, DMSO-d₆) δ 10.11 (1H, s), 8,67 (1H, s), 8.18 (1H,d), 8.08 (2H, d), 7.85 (1H, d), 7.59-7.69 (4H, m), 7.46-7.54 (2H, m),7.35 (2H, t), 4.07 (2H, s); ESI MS m/z 439 (M+H⁺)

Example 2

[0103] 4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0104] A solution of 4-chloro-quinoline-3-carboxylic acid ethyl ester(11.8 mg, 0.5 mmol) and 4-hydrazino-benzoic acid (7.6 mg, 0.5 mmol) inn-butanol (0.5 mL) was stirred at 115° C. over night in a sealed tube.After cooling to 50-70° C., heptane (1.0 mL) was added and the productwas allowed to crystallize upon further cooling to room temperature. Thesolvent was removed and the product was washed with heptane and driedunder vacuum to yield4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid (13 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 12.9 (1H, s), 8.77 (1H, d), 8.38 (2H, d),8.25 (1H, d), 8.03 (2H, d), 7.65-7.77 (2H, m), 7.58 (1H, t).

[0105] Using essentially the same procedure the following compounds wereprepared:

[0106] 3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0107]¹H NMR (400 MHz, DMSO-d₆) δ 12.87 (1H, bs), 8.78 (1H, s), 8.72(1H, d), 8.46 (1H, d), 8.23 (1H, d), 7.63-7.72 (3H, m), 7.51-7.56 (2H,m); ESI MS m/z 306 (M+H⁺).

[0108]2-Chloro-4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0109] In this reaction the hydrochloride salt of2-chloro-4-hydrazino-benzoic acid was used and therefor Et₃N (2 eq) wasalso added to the mixture. ¹H NMR (400 MHz, DMSO-d₆) δ 13.00 (1H, bs),8.76 (1H, d), 8.44 (1H, s), 8.22-8.28 (2H, m) , 7.94 (1H, d) 7.65-7.74(2H, m), 7.55 (1H, t); ESI MS m/z 340 (M+H⁺).

[0110]4-(6-Methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0111]¹H NMR (400 MHz, DMSO-d₆) δ 12.1 (1H, s), 8.52 (1H, d), 8.38 (2H,d), 8.12 (1H, d), 8.03 (2H, d), 7.56 (1H, d), 7.48 (1H, t) , 2.58 (3H,s).

[0112]4-(8-Methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0113]¹H NMR (400 MHz, DMSO-d₆) δ 12.9 (1H, s), 8.73 (1H, d) 8.39 (2H,d), 8.06 (1H, s), 8.03 (2H, d), 7.64 (1H, d), 7.53 (1H, d), 2.50 (3H, s,in DMSO signal).

[0114]4-(6,8-Dimethyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0115]¹H NMR (400 MHz, DMSO-d₆) δ 8.48 (1H, s), 8.38 (2H, d), 8.03 (2H,d), 7.92 (1H, s), 7.40 (1H, s), 2.57 (3H, s), 2.45 (3H, s).

[0116]4-(8-tert-Butyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0117]¹H NMR (400 MHz, DMSO-d₆) δ 12.9 (1H, s), 8.75 (1H, d) , 8.40 (2H,d), 8.17 (1H, s), 8.03 (2H, d), 7.81 (1H, d), 7.69 (1H, d), 1.40 (9H,s).

[0118]4-(8-Methoxy-6-methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0119]¹H NMR (400 MHz, DMSO-d₆) δ 12.3 (1H, s), 8.45 (1H, d), 8.41 (2H,d), 8.03 (2H, d), 7.49 (1H, d), 7.21 (1H, d), 3.93 (3H, s), 2.57 (3H,s).

[0120]4-(8-Methoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0121]¹H NMR (400 MHz, DMSO-d₆) δ 12.9 (1H, s), 8.71 (1H, d), 8.41 (2H,d), 8.03 (2H, d), 7.70 (1H, d), 7.61 (1H, d), 7.32 (1H, dd), 3.94 (3H,s).

[0122]4-(6,8-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0123]¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (2H, d) , 8.31 (1H, s) 8.03 (2H,d), 7.18 (1H, d), 6.93 (1H, d), 4.03 (3H, s), 3.95 (3H, s).

[0124]4-(7,9-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid

[0125]¹H NMR (400 MHz, DMSO-d₆) δ 12.6 (s, 1H) , 8.61 (1H, s) 8.36 (2H,d), 8.01 (2H, d), 6.79 (1H, d), 6.69 (1H, d), 4.00 (3H, s), 3.87 (3H,s).

[0126]4-(3-oxo-8-trifluoromethyl-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid (60% Pure by NMR)

[0127]¹H NMR (400 MHz, DMSO-d₆) δ 13.1 (1H, s), 8.85 (1H, d) 8.38 (2H,d), 8.10 (1H, s), 8.03 (2H, d), 7.87 (1H, d), 7.73 (1H, d).

[0128]4-(6-Fluoro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0129]¹H NMR (400 MHz, DMSO-d₆) δ 13.0 (1H, s), 8.58 (1H, s), 8.37 (2H,d), 8.06 (1H, d), 8.04 (2H, d), 7.54-7.66 (2H, m).

[0130]4-(6-Chloro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0131]¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (1H, s), 8.36 (2H, d), 8.24 (1H,d), 8.04 (2H, d), 7.88 (1H, d), 7.57 (1H, t).

[0132]4-(8-Chloro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid(85% Pure by NMR)

[0133]¹H NMR (400 MHz, DMSO-d₆) δ 13.0 (1H, s), 8.82 (1H, d), 8.38 (2H,d), 8.20 (1H, s), 8.03 (2H, d), 7.76 (2H, s).

[0134]4-(7,9-Dichloro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicAcid (60% Pure by NMR)

[0135]¹H NMR (400 MHz, DMSO-d₆) δ 13.0 (s, 1H), 8.81 (1H, s), 8.36 (2H,d), 8.04 (2H, d), 7.78 (1H, d), 7.71 (1H, d).

Example 3

[0136][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-propionicAcid

[0137] A suspension of 4-chloro-quinoline-3-carboxylic acid ethyl ester(0.050 g, 0.21 mmol) and 3-(4-hydrazino-phenyl)-propionic acid (0.046 g,0.25 mmol) in 2-propanol (3 ml) was heated at 84° C. in an oil bath for10 h. The precipitate was collected, washed with 2-propanol and dried toafford the 2-propylester of the product (0.031 g). ¹H NMR (400 MHz,DMSO-d₆) δ 12.8 (1H, bs) , 8.66 (1H, d) , 8.18 (1H, d) , 8.07 (2H, d),7.60-7.72 (2H, m) , 7.52 (1H, t) , 7.26 (2H, d) , 4.85 (1H, m) , 2.82(2H, t) , 2.55 (2H, t) , 1.12 (6H, d).

[0138] The ester (0.019 g) was hydrolysed by dissolving it in EtOH (0.8ml) and 1M NaOH (0.4 ml). After 90 minutes the mixture was acidifiedwith 2 M HCl and the precipitate was collected, washed with water anddried to yield[4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-propionicacid (0.016 g): ¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (1H, s), 8.18 (1H, d),8.07 (2H, d), 8.61-8.69 (2H, m), 7.51 (1H, t), 7.26 (2H, d), 2.81 (2H,t), 2.52 (2H, t) ; ESI MS m/z 334 (M+H⁺).

[0139] Using essentially the same procedure the following compounds wereprepared:

[0140][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-acetic Acidn-butyl Ester

[0141]¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (1H, bs) , 8.68 (1H, d) 8.18 (1H,d), 8.13 (2H, d), 7.61-7-72 (2H, m), 7.53 (1H, t), 7.30 (2H, d), 4.02(2H, t), 3.63 (2H, s), 1.52 (2H, m) , 1.28 (2H, m) , 0.83 (3H, t) ; and

[0142][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-acetic Acid

[0143]¹H NMR (400 MHz, DMSO-d₆) δ 8.67 (1H, s), 8.19 (1H, d), 8.11 (2H,d), 7.61-7.69 (2H, m), 7.52 (1H, t), 7.29 (2H, d), 3.54 (2H, s); ESI MSm/z 320 (M+H⁺).

Example 4

[0144] 3-(4-Hydrazino-phenyl)-propionic Acid

[0145] A suspension of 3-(4-aminophenyl)-propionic acid (0.50 g, 3.0mmol) in concentrated hydrochloric acid (3.5 ml) was treated with sodiumnitrite (0.21 g, 3.0 mmol) in H₂O (1.7 ml) at 0° C. The reaction wasstirred for 45 minutes after which stannous chloride (1.26 g, 6.7 mmol)in concentrated hydrochloride acid (1.5 ml) was added dropwise at 0° C.The reaction was stirred for 1 h at room temperature. The precipitatewas collected and dried to afford the hydrochloride salt (0.54 g). Thesalt (0.100 g) was dissolved in a small amount of water and made basicwith 1M NaOH. The solid material was removed by filtration and thefiltrate was acidified with acidic acid to yield a precipitate. Thesolid was collected, washed with water and dried to afford3-(4-hydrazino-phenyl)-propionic acid (0.050 g): ¹H NMR (400 MHz,DMSO-d₆) δ 6.90 (2H, d) , 6.64 (2H, d) , 2.63 (2H, t) , 2.39 (2H, t).

[0146] Using essentially the same procedure the following compound wasprepared:

[0147] 2-Chloro-4-hydrazino-benzoic Acid Hydrochloride

[0148] In this reaction the hydrochloride salt was collected andpurified by recrystallization from ethanol: ¹H NMR (400 MHz, DMSO-d₆) δ8.97 (1H, bs) , 7.76 (1H, d) , 7.03 (1H, s) , 6.88 (1H, d).

Example 5

[0149]4-[3-(2-Hydroxy-phenyl)-5-oxo-4,5-dihydro-pyrazolo-1-yl]-benzonitrile

[0150] A suspension of 4-hydroxycoumarine (0.71 g, 4.4 mmol) and4-cyanophenylhydrazine (0.88 g, 6.6 mmol) in dry toluene was heated at125° C. in an oil bath. Toluene was slowly distilled off during thereaction to separate water. A total of 30 ml toluene was removed. After3 h the solution was allowed to cool and the precipitate was filtered,washed with toluene and dried. The crude product was dissolved inCH₂Cl₂. The solid material was removed by filtration before the solutionwas washed with 2M HCl. The organic phase was dried, filtrated and thesolvent evaporated to yield4-[3-(2-hydroxy-phenyl)-5-oxo-4,5-dihydro-pyrazolo-1-yl]-benzonitrile(0.44 g): ¹H NMR (400 MHz, CDCl₃) δ 9.86 (1H, s), 8.01 (2H, d), 7.73(2H, d), 7.41 (1H, t), 7.23-7.26 (1H, m), 7.08 (1H, d), 6.98 (1H, t),3.99 (2H, s).

Example 6

[0151] 4-(4-Methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzonitrile

[0152] A mixture of4-[3-(2-hydroxy-phenyl)-5-oxo-4,5-dihydro-pyrazolo-1-yl]-benzonitrile(0.25 g, 0.9 mmol) and triethyl orthoacetate (1.6 ml) was heated at 120°C. in an oil bath for 15 minutes. After cooling the precipitate wasfiltered, washed with diethyl ether and dried to yield4-(4-methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzonitrile (0.22 9):¹H NMR (400 MHz, CDCl₃) δ 8.35 (2H, d), 8.18 (1H, d), 7.70 (2H, d),7.58-7.62 (1H, m), 7.47-7.51 (2H, m), 2.84 (3H, s); ESI MS m/z 302(M+H⁺)

Example 7

[0153] 4-(4-Methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzoic Acid

[0154] A mixture of4-(4-methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzonitrile (0.030 g,0.10 mmol), acetic acid (0.4 ml), H₂SO₄ (0.4 ml) and water (0.4 ml) washeated at 100° C. in an oil bath for 19 h. After cooling water was addedand the precipitation collected, washed with water and dried to yield4-(4-methyl-3-oxo-3H-chromeno-[4,3-c]pyrazol-2-yl)-benzoic acid (0.029g): ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (2H, d), 8.13 (1H, d), 8.01 (2H,d), 7.67-7.75 (2H, m), 7.54-7.58 (1H, m), 2.78 (3H, s); ESI MS m/z 321(M+H⁺).

[0155] Using essentially the same procedure the following compound wasprepared:

[0156] 4-(3-Oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)benzoic Acid:

[0157]¹H NMR (400 MHz, DMSO-d₆) δ 9.29 (1H, s), 8.44 (1H, d), 8.24 (2H,d), 8.03 (2H, d), 7.98 (1H, d), 7.67-7.76 (2H, m); ESI MS m/z 323(M+H⁺).

Example 8

[0158] 4-Oxo-thiochroman-3-carboxylic Acid Methyl Ester

[0159] LHMDS (1.12 g, 6.7 mmol) dissolved in anhydrous THF (7 ml) wascooled to −78° C. under N₂. Thiochroman-4-one (1.00 g, 6.1 mmol) inanhydrous THF (20 ml) was added dropwise under 20 minutes. After anadditional 60 minutes methyl cyanoformate (0.62 g, 7.3 mmol) inanhydrous THF (1.5 ml) was added dropwise under 5 minutes and thesuspension was then stirred at −78° C. for 80 minutes. The suspensionwas poured onto 10% NH₄Cl and extracted with ether. The organic phasewas washed with water, dried (Na₂SO₄), filtered and the solventevaporated. The crude product was purified by chromatography on silicagel (heptane-ethyl acetate 10:1) to yield 4-oxo-thiochroman-3-carboxylicacid methyl ester (0.65 g): ¹H NMR (400 MHz, CDCl₃; enol tautomer) δ12.64 (1H, s), 7.83 (1H, d), 7.25-7.29 (2H, m), 7.16-7.20 (1H, m), 3.84(3H, s), 3.71 (2H, s).

Example 9

[0160]4-(3-Oxo-1,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzonitrile

[0161] 4-Oxo-thiochroman-3-carboxylic acid methyl ester (0.200 g, 0.90mmol), 4-cyanophenylhydrazine (0.132 g, 0.99 mmol) together with a smallamount of pivalic acid was heated at 118° C. in an oil bath under N₂.After 1 h the mixture was cooled to room temperature and then trituratedwith ether. The precipitate was filtered and dried to yield4-(3-oxo-1,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzonitrile(0.233 g): ¹H NMR (400 MHz, DMSO-d₆) δ 8.06 (2H, d), 7.92 (2H, d),7.80-7.83 (1H, m), 7.30-7.33 (1H, m), 7.18-7.25 (2H, m), 3.91 (2H, s).

Example 10

[0162] 4-(3-Oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzonitrile

[0163] A solution of4-(3-oxo-1,4-dihydro-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzonitrile(0.100 g, 0.33 mmol) in DMSO (2 ml) was stirred vigorously at roomtemperature and air was flushed over the solution. After 48 h theprecipitation was filtered, washed with toluen and dried. The crudeproduct recrystallized from toluene to yield4-(3-oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)-benzonitrile (0.036 g): ¹HNMR (400 MHz, CDCl₃) δ 8.65 (1H, s), 8.53 (1H, d), 8.38 (2H, d), 7.72(2H, d), 7.66 (2H, t), 7.58-7.62 (1H, m); ESI MS m/z 304 (M+H⁺).

Example 11

[0164] 4-(5-Methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic Acid

[0165] A suspension of4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid (0.050g, 0.16 mmol) in DMF (1 ml) was added in small portions to a suspensionof NaH (55%) (0.017 g, 0.39 mmol) in DMF (0.5 ml) under N₂. The reactionwas stirred at room temperature. After 1 h MeI (0.053 g, 0.38 mmol) wasadded. After additional 24 h water was added and the solid material(mainly consisting of ester product) was removed by filtration. Thefiltrate was acidified with 2M HCl. The precipitate was collected,washed with water and dried to yield4-(5-methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid(0.018 g). This material contained approx. 10% of ester product: ¹H NMR(400 MHz, DMSO-d₆) δ 8.89 (1H, s), 8.34 (2H, d), 8.29 (1H, d), 7.99 (2H,d), 7.85 (1H, d), 7.76 (1H, t), 7.62 (1H, t), 4.00 (3H, s); ESI MS m/z320 (M+H⁺).

Example 12

[0166] 2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-3-phenyl-propionic Acid

[0167] 4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid(30 mg, 0.1 mmol) was dissolved in DMF (0.5 ml) and 18 mg (0.1 mmol) ofcarbonyldiimidazol (90% pure) was added and the mixture stirred for 4 h.Phenylalanine (33 mg, 0.2 mmol) was dissolved in of H₂O (0.5 ml)together with triethylamine (0.05 ml) and added to the activated acid.This reaction mixture was heated at 75° C. for 2 h. After cooling it wasmade acidic and diluted 5×with H₂O. The precipitate was filtered andwashed with H₂O and dried under vacuum. FlashTube chromatography usingEtOAc/MeOH(7:3) yielded a small amount of2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-3-phenyl-propionic acid. The material contained approx.10% of starting material. ¹H NMR (400 MHz, DMSO-d₆) δ 8.7 (1H, s), 8.31(2H, d), 8.21 (1H, d), 7.82 (2H, d), 7.70 (1H, d), 7.62 (1H, t), 7.50(1H, t), 7.2-7.3(4H, m), 7.17 (1H, t) , 4.50 (1H, m) , 3.1-3.2 (2H,m).

[0168] Using essentially the same procedure the following compound wasprepared:

[0169] 2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-2-acetic Acid

[0170]¹H NMR (400 MHz, DMSO-d₆) δ 8.7 (1H, s) , 8.36 (2H, d), 8.22 (1H,d) , 7.92 (2H, d) , 7.70 (1H, d) , 7.63 (1H, t) 7.51 (1H, t) , 3.85(2H,d)

Example 13

[0171] Time resolved fluorescence competition assay protocol:

[0172] CD28-CD80 interactions. Reagent Supplier Eu-labelled anti-RabbitWallac Oy, Turku, Finland, antibody (αR-Eu) Rabbit anti-mouse IgG,Jackson Immunoresearch Fc fragment specific Laboratories Inc., (RαmIg(Fc)) Human CD28-mouse Active Biotech Research AB IgG1 (Fc) (CD28-mFc)Human CD80-mouse C215 Active Biotech Research AB Fab (C2l5Fab-hCD80)Mouse C215Fab Active Biotech Research AB Biotin conjugated Goat SouthernBiotechnology Ass. anti-mouse IgG Kappa Inc. light chain (GαMk-biot)Streptavidin- Wallac Oy, Turku, Finland allophycocyanin (SA-APC)

[0173] Assay Buffer

[0174] 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8, containing0.1% BSA (w/v), added prior to use.

[0175] Sample Preparation

[0176] The substances to be tested for inhibitory effects were seriallydiluted from stock concentrations of 20 mM (DMSO) in assay buffer topreparation concentrations of 200, 100, 50 and 25 μM. The finalconcentration in the wells was 100, 50, 25 and 12.5 μM, respectively.The maximum final DMSO concentration tolerated in the assay was 0.5%.Where appropriate, the dilution series was adjusted and extended tomeasure the IC₅₀.

[0177] Control

[0178] CTLA4-hIg(Fc) was diluted to working concentrations of 20 nM and2 nM, resulting in final concentrations of 10 nM and 1 nM in the well.At these concentrations (10 nM and 1 nM) approximately 90% and 30%inhibition levels were observed.

[0179] Preparation of Reagent Mixture

[0180] For 1 plate:

[0181] To a tube containing 1.1 ml assay buffer, the following reagentswere added: Reagent Volume/1.1 ml Preparation Final conc assay bufferconc in well αR-Eu  4.2 μl from 0.528 2 μg/ml 1 μg/ml mg/ml RαmFc  2.8μl from 2.4 6 μg/ml 3 μg/ml mg/ml CD28-  2.6 μl from 0.4 0.95 μg/ml 0.48μg/ml mFc mg/ml (10 nM) (5 nM) Gαmκ-  8.8 μl from 0.5 4 μg/ml 2 μg/mlbiotin mg/ml SA-APC 15.4 μl from 1 16 μg/ml 8 μg/ml mg/ml

[0182] CD80FabC215 Mixture

[0183] 900 μl of the above reagent mixture was transferred to a newtube. CD80FabC215 was added at a preparation concentration of 20 nM,(i.e. 4.3 μl from 0.4 mg/ml stock). The final concentration in the wellwas 10 nM.

[0184] FabC215 Mixture

[0185] To the remaining 200 μl reagent mixture C215Fab was added ascontrol for non-specific binding (NSB). 0.5 μl from a 0.36 mg/ml stockgave a preparation concentration of 20 nM and a final concentration inthe well of 10 nM.

[0186] Pipetting

[0187] To a black, half area, 96-well microtiter plate, the C215Fabcontaining reagent mixture was transferred to column 12, row E-H (4wells), 10 μl/well with a single channel electronic pipette. 10 μl/wellof the CD80FabC215 containing reagent mixture was transferred with asingle channel electronic pipette to all other wells on the plate

[0188] B0: Assay buffer, 10 μl/well was pipetted to the CD80FabC215containing wells in column 12, row A-D (4 wells).

[0189] NSB: Assay buffer, 10 μl/well was pipetted to the C215Fabcontaining wells in column 12, row E-H (4 wells).

[0190] CTLA4-hIg(Fc), 10 μl/well of 20 and 2 nM (final conc 10 and 1 nM)was pipetted in duplicate into well 11 E-F and 11 G-H.

[0191] Sample dilution series: 10 μl/well in duplicate to the remainingwells.

[0192] Incubation

[0193] The plate was covered with a plastic lid and incubated in thedark: Initially for 1 h on a shaker platform at room temperature (RTS),stationary at +4° C. overnight, and finally 1 h RTS before reading.

[0194] Measurement

[0195] The plate was measured on a Victor 1420 Multilabel Counter usingthe LANCE protocol (#2) measuring emission at dual wavelengths, fromboth APC (665 nm) and Europium (615 nm). First measurement: Excitation340 nm, emission 665 nm, delay 50 μs, window time 200 μs. Secondmeasurement: Excitation 340 nm, emission 615 nm, delay 50 μs, windowtime 200 μs.

[0196] Calculation

[0197] The fluorescence signal ratio 1000* 665 nm/615 nm, from which thepercentage inhibition was calculated, was determined. A logit b plot(Logit b=LN (% Bound/(100% −% Bound, plotted against Log conc) wasperformed from which IC₅₀ was measured.

[0198] The assay was performed at least twice in order to have twocomparable IC₅₀ values of the compounds.

[0199] Results

[0200] The following representative results were obtained:

[0201] 4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid

[0202] IC₅₀=0,48 μM

[0203]2-Chloro-4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid

[0204] IC₅₀=0,27 μM

[0205] 3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoic acid

[0206] IC₅₀=0,88 μM

[0207]N-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicacid

[0208] IC₅₀=0,6 μM

[0209][4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-propionicacid

[0210] IC₅₀=1,6 μM

[0211] 2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-2-acetic acid

[0212] IC₅₀=3,9 μM

[0213] 4-(3-Oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)benzoic acid

[0214] IC₅₀=13,5 μM

1. A compound having the general formula (I)

wherein X represents a bond or a group selected from substituted orunsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ or C(O)—NH— (amino acid residue); Y represents NR4, O or S; R1 represents H,halo, CF₃, lower alkyl or lower alkoxy; R2 and R4 representsindependently H or lower alkyl; and R3 represents h, halo, lower alkylor lower alkoxy, wherein halo is F, Cl or Br; wherein lower alkylrepresents saturated or unsaturated, straight, branched or cyclic alkylgroups having 1-6 carbon atoms; and wherein lower alkoxy representssaturated or unsaturated, straight, branched or cyclic alkoxy groupshaving 1-6 carbon atoms, with the proviso that R2 is not H, when X is abond, Y is NH and R3 is H, or pharmaceutically acceptable salts thereof:2. A compound according to claim 1, wherein X is a bond.
 3. A compoundaccording to claim 1 or 2, wherein Y is NH.
 4. A compound according toany one of claims 1-3, selected from the group comprising{[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy}-aceticacid,N-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicacid,4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-butyricacid,{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methoxy}-aceticacid,4-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-2-phenyl-butyricacid,N-[3-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-succinamicacid,2-{[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenylcarbamoyl]-methyl}-benzoicacid,2-Chloro-4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(6,8-Dimethyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(8-Methoxy-6-methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(6,8-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(7,9-Dimethoxy-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(6-Methyl-3-oxo-8-trifluoromethyl-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,4-(7,9-Dichloro-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-propionicacid, [4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-phenyl]-aceticacid, 4-(4-Methyl-3-oxo-3H-chromeno[4,3-c]pyrazol-2-yl)-benzoic acid,4-(3-Oxo-3H-thiochromeno[4,3-c]pyrazol-2-yl)benzoic acid,4-(5-Methyl-3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoicacid,2-[4-(3-oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-3-phenyl-propionicacid, and2-[4-(3-Oxo-3,5-dihydro-pyrazolo[4,3-c]quinolin-2-yl)-benzoylamino]-2-aceticacid.
 5. A compound according to any one of claims 1-4, which is a CD80antagonist, capable of inhibiting the interaction between CD80 and CD28.6. A compound according to any one of claims 1-5 for use as amedicament.
 7. A compound according to claim 6 for use as a medicamentfor treatment of medical conditions chosen from the group comprisingrheumatoid arthritis, multiple sclerosis, diabetes, asthma,transplantation, systemic lupus erythematosis and psoriasis.
 8. Use of acompound as defined in any one of claims 1-5 as a prodrug, preferably inthe form of an ester.
 9. A pharmaceutical composition comprising acompound according to any one of claims 1-6 as active ingredient inassociation with a pharmaceutically acceptable adjuvant, diluent orcarrier.
 10. A pharmaceutical composition according to claim 9 fortreatment of medical conditions chosen from the group comprisingrheumatoid arthritis, multiple sclerosis, diabetes, asthma,transplantation, systemic lupus erythematosis and psoriasis.
 11. Use ofa compound having the general formula (I)

wherein X represents a bond or a group selected from substituted orunsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ or C(O)—NH— (amino acid residue); Y represents NR4, O or S; R1 represents H,halo, CF₃, lower alkyl or lower alkoxy; R2 and R4 representsindependently H or lower alkyl; and R3 represents H, halo, lower alkylor lower alkoxy, wherein halo is F, Cl or Br; wherein lower alkylrepresents saturated or unsaturated, straight, branched or cyclic alkylgroups having 1-6 carbon atoms; and wherein lower alkoxy representssaturated or unsaturated, straight, branched or cyclic alkoxy groupshaving 1-6 carbon atoms, or pharmaceutically acceptable salts thereof,for the manufacture of a medicament for treatment of medical conditionswhich benefit from immunomodulation.
 12. Use according to claim 11,wherein said medical conditions are chosen from the group comprisingrheumatoid arthritis, multiple sclerosis, diabetes, asthma,transplantation, systemic lupus erythematosis and psoriasis.
 13. Amethod for treatment of medical conditions which benefit fromimmunomodulation comprising administration of a therapeuticallyeffective amount of a compound having the general formula (I)

wherein X represents a bond or a group selected from substituted orunsubstituted C₁₋₃-alkyl, NH—C(O)—C₁₋₃-alkyl, NH—C(O)—CH₂—O—CH₂ or C(O)—NH— (amino acid residue); Y represents NR4, O or S; R1 represents H,halo, CF₃, lower alkyl or lower alkoxy; R2 and R4 representsindependently H or lower alkyl; and R3 represents H, halo, lower alkylor lower alkoxy, wherein halo is F, Cl or Br; wherein lower alkylrepresents saturated or unsaturated, straight, branched or cyclic alkylgroups having 1-6 carbon atoms; and wherein lower alkoxy representssaturated or unsaturated, straight, branched or cyclic alkoxy groupshaving 1-6 carbon atoms, or pharmaceutically acceptable salts thereof.14. A method according to claim 13, wherein said medical conditions arechosen from the group comprising rheumatoid arthritis, multiplesclerosis, diabetes, asthma, transplantation, systemic lupuserythematosis and psoriasis.
 15. A method according to claim 13 or 14,wherein said therapeutically effective amount per day is within therange of 0,001-10 mg/kg body weight, preferably within the range of0,1-5 mg/kg body weight.